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Enhanced CRISPR-Based DNA Demethylation by Casilio-ME-Mediated RNA-Guided Coupling of Methylcytosine Oxidation and DNA Repair Pathways

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Enhanced CRISPR-Based DNA Demethylation by Casilio-ME-Mediated RNA-Guided Coupling of Methylcytosine Oxidation and DNA Repair Pathways ARTICLE https://doi.org/10.1038/s41467-019-12339-7 OPEN Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways Aziz Taghbalout1, Menghan Du1, Nathaniel Jillette1, Wojciech Rosikiewicz1, Abhijit Rath2, Christopher D. Heinen2, Sheng Li1 & Albert W. Cheng 1,3,4* Casilio-ME 1234567890():,; Here we develop a methylation editing toolbox, , that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boost gene activation and methylcytosine demethylation of targeted loci. We expand the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes. 1 The Jackson Laboratory for Genomic Medicine, 10 Discovery Drive, Farmington, CT 06032, USA. 2 Center for Molecular Oncology, University of Connecticut Health, 263 Farmington Avenue, Farmington, CT 06030, USA. 3 Department of Genetics and Genome Sciences, University of Connecticut Health, 400 Farmington Avenue, Farmington, CT 06030, USA. 4 Institute for Systems Genomics, UConn Health Science Center, 400 Farmington Avenue, Farmington, CT 06030, USA. *email: [email protected] NATURE COMMUNICATIONS | (2019) 10:4296 | https://doi.org/10.1038/s41467-019-12339-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-12339-7 NA methylation is part of the multifaceted epigenetic mediating transcriptional activation of methylation-silenced Dmodifications of chromatin that shape cellular differ- gene and 5mC demethylation. The ability of Casilio-ME to entiation, gene expression, and maintenance of cellular mediate co-delivery of TET1 activity along with other protein homeostasis. Aberrant DNA methylation is implicated in various factors, which enhance turnover of oxidized cytosine inter- diseases including cancer, imprinting disorders, and neurological mediates, paves the way for new areas of research to efficiently diseases1. Developing tools to directly edit the methylation state address the cause–effect relationships of DNA methylation in of a specific genomic locus is of significant importance both for normal and pathological processes. studying the biology of DNA methylation as well as for devel- opment of therapies to treat DNA methylation-associated diseases. Results In mammalian cells, the 5-methylcytosine (5mC) epigenetic Casilio-ME1 delivers TET1 activity to a genomic target site. mark generated by covalent linkage of a methyl group to the 5th Casilio-ME1 is a three-component DNA Methylation Editing position of the cytosine ring of CpG sequences is catalyzed by one platform built on Casilio which uses nuclease-deficient Cas9 of the three canonical DNA methyltransferases DMNT1, (dCas9), an effector module made of Pumilio/FBF (PUF) – DNMT3A, and DNMT3B2 4. DNA methylation is dynamic and domain linked to an effector protein, and a modified sgRNA involves demethylation pathways which erase 5mC to restore containing PUF-binding sites (PBS) (Fig. 1a)30. The dCas9/ unmethylated DNA. Active demethylation involves the ten–eleven sgRNA ribonucleoprotein complex binds DNA targets without translocation (TET) family of methylcytosine dioxygenases that cutting to serve as an RNA-guided DNA-binding vehicle whose iteratively oxidize 5mC into 5-hydroxymethylcytosine (5hmC), 5- specificity is dictated by the spacer sequence of the sgRNA and formylcytosine (5fC), and 5-carboxylcytosine (5caC) intermediates5. a short protospacer adjacent motif located on the target DNA. Subsequently, 5fC and 5caC are processed by the base-excision PUF-tethered effectors are recruited to the ribonucleoprotein repair (BER) machinery to restore unmethylated cytosines. complex via binding to their cognate PBS present on the sgRNA Restoration of an intact DNA base is initiated by DNA glycosylases scaffold. PUF domains are found in members of an evolutio- that excise damaged bases to generate an apurinic/apyrimidinic site narily conserved family of eukaryotic RNA-binding proteins (AP site) for processing by the rest of the BER machinery. Thymine whose specificity is encoded within their structural tandem DNA glycosylase (TDG)-based BER has been functionally linked to repeats, each of which recognizes a single ribonucleobase31. TET1-mediated demethylation, suggesting an interplay between PUF domains can be programmed to bind to any 8-mer RNA TET1 and enzymes of the BER machinery to actively erase 5mC sequence, e.g., PUFa and PUFc used in this study were designed marks. TDG acts on 5fC and 5caC and NEIL1 and NEIL2 DNA to bind PBSa (UGUAUGUA) and PBSc (UUGAUGUA), glycosylase/AP-lyase activities facilitate restoration of unmethylated respectively30,31. Multiple PBS added in tandem to the 3′ end of cytosine by displacing TDG from the AP site to create a single the sgRNA allow concurrent recruitment of multiple PUF- – strand DNA break substrate for further BER processing6 12. effectors to targeted DNA sequences without interfering with Interestingly, DNA demethylation is enhanced by GADD45A dCas9 targeting, and therefore allow amplification of the (Growth Arrest and DNA-Damage-inducible Alpha), a multifaceted response to associated effector modules30. nuclear protein involved in maintenance of genomic stability, DNA To enable targeted cytosine demethylation and subsequent – repair and suppression of cell growth13 15. GADD45A interacts activation of methylation-silenced genes, we built a DNA methyl with TET1 and TDG, and was suggested to play a role in coupling editor TET1-effector Casilio-ME1 as a protein fusion of hTET1 5mC oxidation to DNA repair16,17. catalytic domain (TET1(CD)) to the carboxyl end of PUFa Advances in artificial transcription factor (ATF) technologies (Fig. 1a). We chose as a target the MLH1 promoter region that is have enabled direct control of gene expression and epigenetic part of a large CGI whose aberrant hypermethylation induces – states18 20. CRISPR/Cas9-based technologies allow much flex- MLH1-silencing in 10–30% of colorectal and other cancers32,33. ibility and scalability because the specificity is programmable by a MLH1 is silenced in HEK293T cells and therefore represents a single guide RNA (sgRNA)21,22. Tethering of TET1 or DNMT3a clinically relevant model for developing Casilio-ME. to genomic targets by use of ATFs has been shown to allow To test the system, cells were transiently transfected with – targeted removal or deposition of DNA methylation23 29. How- plasmids encoding Casilio-ME1 components PUFa-TET1(CD) ever, these ATF systems have inherent limitations in enabling effector, dCas9 and six MLH1-promoter-targeting sgRNAs each multiplexed targeting, effector multimerization or formation of containing five copies of PBSa (Fig. 1a). This resulted in robust protein complexes at the targeted sequence. We recently devel- MLH1 activation as indicated by the obtained fold changes in oped the Casilio system which uses an extended sgRNA scaffold MLH1 mRNA in cells collected on day 3 post-transfection to assemble protein factors at target sites, enabling multi- (Fig. 1b, c upper panel). In contrast, MLH1 activation was not merization, differential multiplexing30, and potentially stoichio- obtained with a non-targeting sgRNA (NT-sgRNA) (Fig. 1b), metric complex formation. indicating that Casilio-ME1-mediated MLH1 activation requires Here we develop an advanced DNA methylation editing specific targeting of the PUFa-TET1(CD) module directed by the technology which allows targeted bridging of TET1 activity to programmable sgRNAs. BER machinery to efficiently alter the epigenetic state of CpG Evidence that the Casilio-ME1-induced activation of MLH1 targets and activate methylation-silenced genes. Casilio-DNA results from TET1-mediated 5mC demethylation came from Methylation Editing (ME) platforms enable targeted delivery of high throughput bisulfite sequencing (BSeq) of MLH1 ampli- the TET1 effector alone (Casilio-ME1) or in association with cons derived from the cells analyzed in Fig. 1b. BSeq showed GADD45A (Casilio-ME2)orNEIL2(Casilio-ME3)toachieve that targeted delivery of the TET1(CD) effector induces a enhanced 5mC demethylation and gene activation. We show profound decrease in CpG methylation frequency within the that Casilio-ME-mediated delivery of TET1 activity to gene MLH1 promoter region (Fig. 1c lower panel). Demethylation promoters induces robust cytosine demethylation within the activity was prominently higher within CpGs neighboring targeted CpG island (CGI) and activation of gene expression. MLH1-sgRNA sites (Fig. 1c lower panel (arrows)), and seemed When systematically compared to other reported methylation to spread away, albeit with relatively reduced activities. These editing systems, Casilio-ME shows superior activities in
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